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Abstract:

The invention provides a method of modulating electrophysiological
activity of an excitable cell. The method involves causing exogenous
expression of a glycine receptor (GlyR) protein in an excitable cell of a
subject. Thereafter, the excitable cell is exposed to an allosteric
modulator of the GlyR protein. Modulation of the exogenous GlyR protein
(an ion channel) in response to the allosteric modulator modulates the
electrophysiological activity of the excitable cell. The method can be
used to control pain in a subject. The invention further provides a
replication-defective HSV vector comprising an expression cassette
encoding a GlyR protein, stocks and pharmaceutical compositions
containing such vectors, and a transgenic animal.

Claims:

1. A method of modulating the electrophysiological activity of an
excitable cell comprising inducing expression of a glycine receptor
(GlyR) protein in an excitable cell of a subject and subsequently
exposing the excitable cell to an allosteric modulator of the GlyR
protein.

2. The method of claim 1, wherein the excitable cell is a neuron.

3. The method of claim 1, which results in the attenuation of pain in the
subject.

4. (canceled)

5. The method of claim 1, wherein expression of the GlyR protein is
induced by introducing a genetic vector into the excitable cell, which
vector comprises a nucleic acid encoding the GlyR protein in operable
linkage to a promoter suitable for expressing the nucleic acid encoding
the GlyR protein within the excitable cell.

7. The method of claim 1, wherein the GlyR protein comprises an alpha2
subunit of GlyR.

8. The method of claim 1, wherein the GlyR protein comprises an alpha3
subunit of GlyR.

9. The method of claim 1, wherein the GlyR protein comprises an alpha4
subunit of GlyR.

10. The method of claim 1, wherein the GlyR protein comprises a beta
subunit of GlyR.

11. The method of claim 1, wherein the GlyR protein comprises a mutein of
a GlyR subunit.

12. The method of claim 11, wherein the mutein is of the alpha1 subunit
of GlyR.

13. The method of claim 11, wherein the mutein converts the GlyR to a
cationic channel.

14. The method of claim 11, wherein the mutein lacks sites for zinc
potentiation or zinc inhibition, anesthetic potentiation or affinity for
ligands.

15. The method of claim 5, wherein the genetic vector is a
replication-defective HSV vector.

16. The method of claim 1, wherein the allosteric modulator is an agonist
of the GlyR protein.

17. The method of claim 16, wherein the agonist is glycine.

18. The method of claim 1, wherein the subject is human.

19.-33. (canceled)

34. The method of claim 2, wherein the neuron is a peripheral neuron.

35. The method of claim 1, wherein the excitable cell is within the
spinal cord or brain.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional
Patent Application No. 60/917,752, filed May 14, 2007 which is
incorporated by reference.

BACKGROUND OF THE INVENTION

[0003] The potential to modulate the electrophysiological response of
excitable cells (such as neurons and muscle cells) could potentially lead
to treatment of neuromuscular conditions, pain, and other disorders
associated with the activity of such cells. However, the administration
of ligands that act on endogenous ion channels poses significant hurdles
because of the potential for widespread side effects due to systemic
delivery. Moreover, agents that act locally (such as silver or capsaicin)
have unwanted side effects and can potentially cause permanent damage.

[0004] Modulation of neuronal activity by expression of a ligand-gated
anionic channel has been shown previously wherein expression of a
glutamate-gated chloride channel (GluCl), a nicotinicoid family receptor
found in invertebrates, was used to silence neurons (Slimko et al., J.
Neurosci., 22, 7373-9 (2002)). GluCl could be selectively activated by
the addition of ivermectin, a high-potency ligand that has little or no
effects on endogenous mammalian ion channels at low concentrations. For
use in vertebrates, and particularly in human patients, however, this
approach poses a risk of generating an immune response against such a
foreign protein, leading to potential autoimmune disorders. Accordingly,
additional methods and reagents for modulating the electrophysiological
activity of excitable cells are desired.

BRIEF SUMMARY OF THE INVENTION

[0005] The invention provides a method and reagents for modulating the
electrophysiological activity of an excitable cell. The method involves
causing exogenous expression of a glycine receptor (GlyR) protein in an
excitable cell of a subject. Thereafter, the excitable cell is exposed to
an allosteric modulator of the GlyR protein. Modulation of the exogenous
GlyR protein (an ion channel) in response to the allosteric modulator
modulates the electrophysiological activity of the excitable cell. The
method can be used to control pain in a subject. The invention further
provides a replication-defective HSV vector comprising an expression
cassette encoding a GlyR protein and stocks and pharmaceutical
compositions containing such vectors. The invention further provides a
transgenic animal comprising an exogenously expressed GlyR protein. These
aspects and other inventive features are further addressed in the
accompanying drawings and the following detailed description.

[0010] FIG. 4B is a graph demonstrating that exogenously applied glycine
reduces formalin-induced nociceptive behavior in rats following injection
of a GlyR-expressing HSV vector, and that alleviation of observed pain
was reversed by strychnine injection.

DETAILED DESCRIPTION OF THE INVENTION

[0011] In one embodiment, the invention provides a method of modulating
the electrophysiological activity of an excitable cell. The method
involves causing exogenous expression of a glycine receptor (GlyR)
protein in an excitable cell of a subject. Thereafter, the excitable cell
is exposed to an allosteric modulator of the GlyR protein. Modulation of
the exogenous GlyR protein (an ion channel) in response to the allosteric
modulator modulates the electrophysiological activity of the excitable
cell.

[0012] The excitable cell can be any cell that experiences fluctuations in
its membrane potential as a result of gated ion channels. Such cells can
include myocytes, neurons, and the like. However, the inventive method is
particularly well suited for application to peripheral neurons.

[0013] The method typically is applied to excitable cells of mammalian
subjects, but can be applied to subjects of other chordate phyla (e.g.,
avians, reptiles, amphibians, bony and cartilaginous fish, etc.). Indeed,
employment of the method in such creatures as well as common laboratory
mammals (e.g., mice, rats, guinea pigs, dogs, monkeys, apes, etc.) can be
useful in biomedical research. Typically, however the method is employed
in connection with excitable cells of mammals, and can be used medically
in human patients (subjects) (e.g., to treat pain). The method also can
be practiced on veterinary patients (subjects) such as cats, dogs, pigs,
horses, cattle, sheep, and the like.

[0014] The inventive method desirably is used to attenuate the sensation
of pain in a subject (preferably a human subject). The pain can be
isolated pain, or the pain can be associated with a particular disease.
The pain can be associated with any known human disease, including but
not limited to, diabetes, arthritis, cardiovascular disease, autoimmune
disease, respiratory disease (e.g., emphysema), infectious disease (e.g.,
viral or bacterial infections), neurological disease (e.g., Alzheimer's
disease), gastrointestinal disease, liver disease, blood disorders,
allergies, endocrine disease, and cancer. The pain can be associated with
cancer of the oral cavity (e.g., tongue cancer and mouth cancer), the
pharynx, the digestive system (e.g., the esophagus, stomach, small
intestine, colon, rectum, anus, liver, gall bladder, and pancreas), the
respiratory system (e.g., lung cancer), bones and joints (e.g., bony
metastases, osteosarcoma), soft tissue, the skin (e.g., melanoma),
breast, the genital system (e.g., ovarian cancer), the urinary system
(e.g., bladder cancer, renal cancer), the eye and orbit, the brain and
nervous system (e.g., glioma), or the endocrine system (e.g., thyroid).
The cancer also can be a lymphoma (e.g., Hodgkin's disease and
Non-Hodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute
lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid
leukemia, chronic myeloid leukemia, and the like).

[0015] The GlyR protein is exogenous in the sense that it is not natively
expressed in the excitable cell to be treated in accordance with the
inventive method. Generally, GlyRs are expressed primarily in cells
within the spinal cord and lower brain. Thus, where even a wild-type GlyR
protein (i.e., other than a mutein) is expressed in, for example,
peripheral neurons, its expression in such cells is exogenous. Also,
exogenous expression can be expression of a GlyR protein at significantly
higher levels than wild-type expression. Thus, inducement of expression
of a GlyR protein in a cell expressing the GlyR protein at a low level is
regarded as "exogenous" if the excitable cell is induced to produce
measurably more GlyR protein as a result of the induction.

[0016] Any suitable method can be employed to cause or induce exogenous
expression of the GlyR protein in the excitable cell. For example, an
agent can be applied to the excitable cell that activates transcription
of the gene encoding the GlyR protein from the genome of the excitable
cell. However, preferably, exogenous expression of the GlyR protein is
caused or induced by gene transfer technology. In this respect, the
method can involve introducing a genetic vector into the excitable cell,
which vector comprises an expression cassette including a nucleic acid
encoding the GlyR protein.

[0017] The GlyR is a member of the nicotinicoid superfamily of
ligand-gated ionotropic receptors that mediate fast neurotransmission in
the central nervous system (CNS). In the case of the GlyR, binding of
glycine (EC50 of 20 μM-100 μM) or other agonists leads to
transient gating of this anion-selective channel. In adults, the GlyR is
believed to typically have a stoichiometry of 2 α subunits and 3
β subunits. Heterologous expression of just the human α1
subunit, however, is sufficient to reconstitute an active glycine-gated
channel with pharmacological properties essentially identical to those of
native channels. Accordingly, for use in the inventive method, the GlyR
protein can be a wild-type subunit of GlyR (e.g., alpha1, alpha2, alpha3,
alpha4, or beta), and is preferably a subunit of mammalian origin or a
mutein of such subunit. The GlyR proteins are well characterized
(Rajendra et al., Pharmacol. Ther., 73(2): 121-46 (1997)) and the
sequences encoding many subunits from mammalian species are indexed in
genetic databases or are otherwise available. For example, sequences
relating to the alpha1 subunit of GlyR can be found at NCBI Accession
Nos. NM--000171 (human), NM--020492 (mouse) and NM--013133
(rat). Sequences relating to the alpha2 subunit of GlyR can be found at
NCBI Accession Nos. NM--002063 (human), CR450343 (cDNA) (human)),
NM--183427 (mouse), and NM--012568 (rat). Sequences relating to
the alpha3 subunit of GlyR can be found at NCBI Accession Nos.
NM--006529 (human), NM--001042543 (human), BC036086 (human),
NM--080438 (mouse), AY230204 (mouse), AF362764 (mouse), and
NM--053724 (rat). Sequences relating to the alpha4 subunit of GlyR
can be found at NCBI Accession Nos. NM 010297 (mouse), and BC110630
(mouse). Sequences relating to the beta subunit of GlyR can be found at
NCBI Accession Nos. NM--000824 (human), NM--010298 (mouse), and
NM--053296 (rat).

[0018] In addition to wild-type GlyR subunits, mutant forms of GlyR
subunit with altered activity (muteins) also are known, and can be used
in the context of the present invention. In this regard, the GlyR protein
can comprise a mutein of any of the GlyR subunits. For example, certain
muteins of GlyR proteins result in altered ion-channel properties, such
as resulting in a cationic ion channel (e.g., Δ250 A251E Keramidas
et al., J. Gen. Physiol., 119, 393 (2002)). Other muteins are known that
lack sites for zinc potentiation or zinc inhibition (Hirzel et al.,
Neuron, 52, 679-90 (2006)), affinity for allosteric modulators (e.g.,
anesthetic potentiation (Hemmings et al., Trends Pharmacol. Sci., 26,
503-10 (2005)), or affinity for ligands (Rajendra et al., Neuron, 14,
169-175 (1995); Schrnieden et al., Science, 262, 256-258 (1993)).
Mutation of GlyR subunits also can selectively alter ion permeation
(e.g., anionic- or cationic-selective channels), and redesign a receptor
subunit's ligand binding pockets to recognize unique pharmacologic
agents. For example, to alter the sensitivity and selectivity of a GlyR
protein for a particular ligand, point mutations can be made in the
GlyRα1 subunit that are expected to shift the dose response curve
to the left or right (i.e., less or more specific to glycine). Other
mutations can alter the sensitivity of a GlyR protein to certain
anesthetics (e.g., ethanol). For example, a mutation in the mouse glycine
α1 receptor subunit in which a methionine (M) at position 287 is
changed to leucine (L) (M297L) results in greatly enhanced sensitivity to
the volatile anesthetic enflurane. Such GlyR muteins can be employed as
the GlyR protein in the context of the present invention.

[0020] Any suitable vector for introducing the expression cassette
encoding the GlyR protein into the excitable cell can be employed.
Examples of suitable vectors include plasmids, liposomes, molecular
conjugates (e.g., transferrin), and viruses. Preferably, the vector is a
viral vector. Suitable viral vectors include, for example, retroviral
vectors, herpes virus based vectors and parvovirus based vectors (e.g.,
adeno-associated virus (AAV) based vectors, AAV-adenoviral chimeric
vectors, and adenovirus-based vectors). Most preferably, the vector is a
replication-defective (also referred to as "replication-deficient")
herpes simplex virus (HSV) vector.

[0021] A vector comprising an expression cassette including a nucleic acid
encoding the GlyR protein can facilitate transfer of the GlyR expression
cassette to the excitable cell by infecting (or transfecting, as
appropriate) the excitable cell with the vector. Given their neurotropic
properties, replication-deficient HSV-based vector systems can be used to
deliver the GlyR expression cassette to the excitable cell by peripheral
inoculation. Following inoculation of a site on the skin, mucus membrane,
or other peripheral site, replication-deficient HSV vectors infect the
excitable cell, which facilitates expression of the GlyR protein within
the infected excitable cell. However, because such vectors are
replication-defective, they do not replicate within the excitable cell to
spread to other areas. In this sense, where replication-defective HSV
vectors are employed to deliver the GlyR gene, a site of inoculation can
be selected to target the treatment to a pre-selected area.

[0022] In further performance of the inventive method, an allosteric
modulator or ligand of the GlyR protein is exposed to the excitable cell
in which the exogenous GlyR protein is exogenously expressed. The
allosteric modulator can, for example, agonize or antagonize (open or
close) the GlyR channel, which in turn alters the electrophysiology of
the excitable cell. A preferred agonist is glycine; however other
suitable agonists of the GlyR protein (e.g., taurine and beta-alanine)
can be employed. Also, a suitable antagonist is strychnine; however other
antagonists can be employed. The allosteric modulator can be delivered
systemically (e.g., parenterally). However, it is preferred for the
allosteric modulator to be administered locally (regionally) to the area
encompassing the excitable cell exogenously expressing the GlyR.
Particularly where replication-defective HSV vectors are employed, the
ability to target specific areas, coupled with the limited distribution
of endogenous GlyRs, permits regional delivery of GlyR-specific
allosteric modulators at concentrations sufficient to activate the
receptor and should not lead to unwanted side effects.

[0023] The introduction of a ligand-gated ionotropic receptor (GlyR) that
can be quickly and reversibly activated or inhibited via addition of
exogenous ligands, thereby allowing the permeation of select ions, can
manipulate the resting potential of excitable cells, and hence their
function. The method can be used in many contexts. In one application,
the inventive method can attenuate the sensation of pain in a patient. In
this application, a vector containing an expression cassette encoding the
GlyR protein (e.g., alpha1 subunit) is employed to cause exogenous
expression of the GlyR protein within peripheral neurons, such as
c-fibers, associated with the sensation of pain. Where a
replication-defective HSV vector is employed, the vector can be injected
into a portion of the subject's skin (e.g., a toe), mucus membrane, or
other desired tissue to introduce the expression cassette encoding the
GlyR protein into the peripheral neurons. Thereafter, glycine or another
agonist of the GlyR protein can be injected into the area of the
inoculation to reduce the sensation of pain associated with the
peripheral neurons. The method can be used to reduce the local or
regional sensation of pain in some subjects (patients) partially or
completely.

[0024] In another embodiment, the inventive method can be used to generate
a transgenic animal that exogenously expresses GlyR protein. The animal
can be any animal that is amenable to modification and analysis via
conventional transgenic technology, such as, for example, a zebrafish,
mouse, rat, or pig. The transgenic animal can be generated using any
suitable method known in the art. Typically and preferably, transgenic
animals are generated using embryonic stem (ES) cell technology. ES cells
are obtained by culturing pre-implantation embryos in vitro under
appropriate conditions (Evans et al., Nature, 292:154-156 (1981); Bradley
et al., Nature, 309: 255-258 (1984); Gossler et al., Proc. Acad. Sci.
(USA), 83: 9065-9069 (1986); and Robertson et al., Nature, 322: 445-448
(1986)). Transgenes can be efficiently introduced into the ES cells by
DNA transfection by a variety of methods known to the art, including
calcium phosphate co-precipitation, protoplast or spheroplast fusion,
lipofectin and DEAE-dextran-mediated transfection. Transgenes can also be
introduced into ES cells by retrovirus-mediated transduction or by
micro-injection. Such transfected ES cells can thereafter colonize an
embryo following their introduction into the blastocoel of a
blastocyst-stage embryo and contribute to the germ line of the resulting
chimeric animal (see, e.g., Jaenisch, Science, 240: 1468-1474 (1988)).
Progeny animals carrying the transgene in their germline can be
identified by Southern blot analysis, by polymerase chain reaction (PCR),
and/or by Northern blot analysis. Transgenic animal technology is further
described in, for example, Pinkett, Transgenic Animal Technology: A
Laboratory Handbook, 2nd Ed., Academic Press (2002), and Hofker et
al., eds., Transgenic Mouse Methods and Protocols (Methods in Molecular
Biology), Human Press (2002)).

[0025] To facilitate the inventive method, the invention provides an HSV
vector comprising an expression cassette encoding a GlyR protein. An HSV
based viral vector is suitable for use as a vector to introduce a nucleic
acid sequence into numerous cell types. The mature HSV virion consists of
an enveloped icosahedral capsid with a viral genome consisting of a
linear double-stranded DNA molecule that is 152 kb. In a preferred
embodiment, the HSV based viral vector is deficient in at least one
essential HSV gene. Of course, the vector can alternatively or in
addition be deleted for non-essential genes. Preferably, the HSV based
viral vector that is deficient in at least one essential HSV gene is
replication-deficient. Most replication-deficient HSV vectors contain a
deletion to remove one or more intermediate-early, early, or late HSV
genes to prevent replication. For example, the HSV vector may be
deficient in a gene selected from the group consisting of: ICP0, ICP4,
ICP22, ICP27, ICP47, and a combination thereof. A preferred HSV vector is
deficient for all immediate early genes except for ICP0. Advantages of
the HSV vector are its ability to enter a latent stage that can result in
long-term DNA expression and its large viral DNA genome that can
accommodate exogenous DNA inserts of up to 25 kb.

[0026] HSV-based vectors and methods for their construction are described
in, for example, U.S. Pat. Nos. 7,078,029, 6,261,552, 5,998,174,
5,879,934, 5,849,572, 5,849,571, 5,837,532, 5,804,413, and 5,658,724, and
International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637,
and WO 99/06583, which are incorporated herein by reference. Preferably,
the HSV vector is "multiply-deficient," meaning that the HSV vector is
deficient in more than one gene function required for viral replication.
The sequence of HSV is published (NCBI Accession No. NC--001806; see
also McGoech et al., J. Gen. Virol, 69 (PT 7), 1531-1574 (1988)), which
may facilitate the generation of desired mutations in designing HSV-based
vectors.

[0027] The HSV vector can be deficient in replication-essential gene
functions of only the early regions of the HSV genome, only the
immediate-early regions of the HSV genome, only the late regions of the
HSV genome, or both the early and late regions of the HSV genome. The HSV
vector also can have essentially the entire HSV genome removed, in which
case it is preferred that at least either the viral inverted terminal
repeats (ITRs) and one or more promoters or the viral ITRs and a
packaging signal are left intact (i.e., an HSV amplicon). The larger the
region of the HSV genome that is removed, the larger the piece of
exogenous nucleic acid sequence that can be inserted into the genome.

[0028] It should be appreciated that the deletion of different regions of
the HSV vector can alter the immune response of the mammal. In
particular, the deletion of different regions can reduce the inflammatory
response generated by the HSV vector. Furthermore, the HSV vector's
protein coat (i.e., one or more envelope proteins) can be modified so as
to decrease the HSV vector's ability or inability to be recognized by a
neutralizing antibody directed against the wild-type protein coat. In
addition, one or more envelope proteins of the HSV vector can be modified
so as to alter the binding specificity, attachment, and/or entry of the
HSV vector to a particular cell. For HSV, such manipulations can include
deletion of regions of any one of the ten glycoproteins (gB through gM)
that make up the HSV envelope (e.g., glycoprotein C and/or glycoprotein
D), or insertions of various native or non-native ligands into portions
of an envelope glycoprotein. Manipulation of the envelope proteins can
broaden the range of cells infected by the HSV vector or enable targeting
of the HSV vector to a specific cell type.

[0029] The HSV vector, when multiply replication-deficient, can include a
spacer element to provide viral growth in a complementing cell line
similar to that achieved by singly replication-deficient HSV vectors. The
spacer element can contain any nucleic acid sequence or sequences which
are of the desired length. The spacer element sequence can be coding or
non-coding and native or non-native with respect to the HSV genome, but
does not restore the replication essential function(s) to the deficient
region. In addition, the inclusion of a spacer element in any or all of
the deficient HSV regions will decrease the capacity of the HSV vector
for large inserts. The production of HSV vectors involves using standard
molecular biological techniques well known in the art.

[0030] When the vector is a replication-deficient HSV, the nucleic acid
sequence encoding the protein (e.g., GlyR protein) can be located in the
locus of an essential HSV gene, such as either the ICP4 or the ICP27 gene
locus of the HSV genome. The insertion of a nucleic acid sequence into
the HSV genome (e.g., the ICP4 or the ICP27 gene locus of the HSV genome)
can be facilitated by known methods, for example, by the introduction of
a unique restriction site at a given position of the HSV genome.

[0031] Replication-deficient HSV vectors are typically produced in
complementing cell lines that provide gene functions not present in the
replication-deficient HSV vectors, but required for viral propagation, at
appropriate levels in order to generate high titers of viral vector
stock. A preferred cell line complements for at least one and preferably
all replication-essential gene functions not present in a
replication-deficient HSV vector. The cell line also can complement
non-essential genes that, when missing, reduce growth or replication
efficiency (e.g., UL55). The complementing cell line can complement for a
deficiency in at least one replication-essential gene function encoded by
the early regions, immediate-early regions, late regions, viral packaging
regions, virus-associated regions, or combinations thereof, including all
HSV functions (e.g., to enable propagation of HSV amplicons, which
comprise minimal HSV sequences, such as only inverted terminal repeats
and the packaging signal or only ITRs and an HSV promoter). The cell line
preferably is further characterized in that it contains the complementing
genes in a non-overlapping fashion with the HSV vector, which minimizes,
and practically eliminates, the possibility of the HSV vector genome
recombining with the cellular DNA. Accordingly, the presence of
replication competent HSV is minimized, if not avoided in the vector
stock, which, therefore, is suitable for certain therapeutic purposes,
especially gene therapy purposes. The construction of complementing cell
lines involves standard molecular biology and cell culture techniques
well known in the art.

[0032] The expression of the nucleic acid sequence encoding the GlyR
protein is controlled by a suitable expression control sequence operably
linked to the nucleic acid sequence. Techniques for operably linking
sequences together are well known in the art.

[0033] An "expression control sequence" is any nucleic acid sequence that
promotes, enhances, or controls expression (typically and preferably
transcription) of another nucleic acid sequence. Suitable expression
control sequences include constitutive promoters, inducible promoters,
repressible promoters, and enhancers. The nucleic acid sequence; encoding
the GlyR protein in the vector can be regulated by its endogenous
promoter or, preferably, by a non-native promoter sequence. Examples of
suitable non-native promoters include the human cytomegalovirus (HCMV)
promoters, such as the HCMV immediate-early promoter (HCMV lEp),
promoters derived from human immunodeficiency virus (HIV), such as the
HIV long terminal repeat promoter, the phosphoglycerate kinase (PGK)
promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long
terminal repeat, mouse mammary tumor virus (MMTV) promoters, the Lap2
promoter, or the herpes thymidine kinase promoter, promoters derived from
SV40 or Epstein Barr virus, and the like. In a preferred embodiment, the
promoter is HCMV IEp. In another embodiment, the expression of the
nucleic acid sequence encoding the GlyR protein can be controlled by a
cell-specific promoter (also referred to as a "tissue-specific"
promoter). A promoter is "cell-specific" if its activity is restricted to
certain cell types (e.g., neurons). The cell-specific promoter can be any
such promoter known in the art. For example, the cell-specific promoter
is a promoter that functions only in A∂-fibers, peptidergic
small unmyelinated C-fibers that project to lamina I and lamina IIo, and
non-peptidergic small to medium unmyelinated C-fibers that project to
lamina IIiC. In addition, a moveable component of the natural viral
latency promoter (LAP2) is active in providing long-term gene expression
in both peripheral and brain neurons in animals and can be coupled with
nerve specific promoter/enhancers. Alternatively, expression of the
nucleic acid sequence encoding the GlyR protein can be controlled by a
chimeric promoter sequence. A promoter sequence is "chimeric" if it
comprises at least two nucleic acid sequence portions obtained from,
derived from, or based upon at least two different sources (e.g., two
different regions of an organism's genome, two different organisms, or an
organism combined with a synthetic sequence). Alternatively, the promoter
can be an inducible promoter, i.e., a promoter that is up- and/or
down-regulated in response to an appropriate signal. For example, an
expression control sequence up-regulated by a pharmaceutical agent is
particularly useful in pain management applications. For example, the
promoter can be a pharmaceutically-inducible promoter (e.g., responsive
to tetracycline). Examples of such promoters are marketed by Ariad
Pharmaceuticals, Inc. (Cambridge, Mass.).

[0034] The nucleic acid sequence encoding the GlyR protein can further
comprise a transcription-terminating region such as a polyadenylation
sequence located 3' of the region encoding the protein. Any suitable
polyadenylation sequence can be used, including a synthetic optimized
sequence, as well as the polyadenylation sequence of BGH (Bovine Growth
Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus),
and the papillomaviruses, including human papillomaviruses and BPV
(Bovine Papilloma Virus).

[0035] After the vector has been created, the vector is purified. Vector
purification to enhance the concentration of the vector in the
composition can be accomplished by any suitable method, such as by
density gradient purification, by chromatography techniques, or limiting
dilution purification. The vector, preferably a replication-deficient HSV
vector, is desirably purified from cells infected with the
replication-deficient HSV vector using a method that comprises lysing
cells infected with the HSV vector and collecting a fraction containing
the HSV vector. The cells can be lysed using any suitable method, such as
exposure to detergents, freeze-thawing, and cell membrane rupture (e.g.,
via French press or microfluidization). The cell lysate then optionally
can be clarified to remove large pieces' of cell debris using any
suitable method, such as gentle centrifugation, filtration, or tangential
flow filtration (TFF). The clarified cell lysate then optionally can be
treated with an enzyme capable of digesting DNA and RNA (a "DNase/RNase")
to remove any DNA or RNA in the clarified cell lysate not contained
within the vector particles.

[0036] Generally, the inventive recombinant HSV is most useful when enough
of the virus can be delivered to a cell population to ensure that the
cells are confronted with a predefined number of viruses. Thus, the
present invention provides a stock, preferably a homogeneous stock,
comprising the inventive HSV vector. The preparation and analysis of HSV
stocks is well known in the art. For example, a viral stock can be
manufactured in roller bottles containing cells transduced with the HSV
vector. The viral stock can then be purified (for example, on a
continuous nycodenze gradient), and aliquotted and stored until needed.
Viral stocks vary considerably in titer, depending largely on viral
genotype and the protocol and cell lines used to prepare them.
Preferably, such a stock has a viral titer of at least about 105
plaque-forming units (pfu), such as at least about 106 pfu or even
more preferably at least about 107 pfu. In still more preferred
embodiments, the titer can be at least about 108 pfu, or at least
about 109 pfu, and high titer stocks of at least about 1010 pfu
or at least about 1011 pfu are most preferred.

[0037] The invention additionally provides a composition comprising the
HSV vector and a carrier. The carrier of the composition can be any
suitable carrier for the vector. The carrier typically will be liquid,
but also can be solid, or a combination of liquid and solid components.
The carrier desirably is a pharmaceutically acceptable (e.g., a
physiologically or pharmacologically acceptable) carrier (e.g., excipient
or diluent). Pharmaceutically acceptable carriers are well known and are
readily available. The choice of carrier will be determined, at least in
part, by the particular vector and the particular method used to
administer the composition. The composition can further comprise any
other suitable components, especially for enhancing the stability of the
composition and/or its end-use. Accordingly, there is a wide variety of
suitable formulations of the composition of the invention. The following
formulations and methods are merely exemplary and are in no way limiting.

[0038] Formulations suitable for local (regional) injection or parenteral
administration include aqueous and non-aqueous, isotonic sterile
injection solutions, which can contain anti-oxidants, buffers,
bacteriostats, and solutes that render the formulation isotonic with the
blood of the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The formulations can be presented
in unit-dose or multi-dose sealed containers, such as ampules and vials,
and can be stored in a freeze-dried (lyophilized) condition requiring
only the addition of a sterile liquid excipient, for example, water, for
injections, immediately prior to use. Extemporaneous injection solutions
and suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.

[0039] In addition, the composition can comprise additional therapeutic or
biologically-active agents. For example, therapeutic factors useful in
the treatment of a particular indication can be present. Factors that
control inflammation, such as ibuprofen or steroids, can be part of the
composition to reduce swelling and inflammation associated with in vivo
administration of the vector and physiological distress. Immune system
suppressors can be administered with the composition method to reduce any
immune response to the vector itself or associated with a disorder.
Alternatively, immune enhancers can be included in the composition to
upregulate the body's natural defenses against disease. Antibiotics,
i.e., microbicides and fungicides, can be present to reduce the risk of
infection associated with gene transfer procedures and other disorders.

[0040] The following examples further illustrate the invention but, of
course, should not be construed as in any way limiting its scope.

Example 1

[0041] This example demonstrates targeted delivery of glycine receptors to
peripheral neurons as treatment for pain.

[0042] Production of recombinant virus. The plasmid pSHB2-GlyR was created
by cloning the GlyR sequences into plasmid SHB2 at the BamHI site of the
polylinker. SHB2 was generated by ligation of a HCMV-BGHpA expression
construct from pRC-CMV into plasmid pSASB3 at the unique BamHI site.
pSASB3 was constructed by cloning the Sph I to Afl III (Sal I linkered)
fragment (1928 bp) of the HSV-1 KOS strain genome (nucleotides
124485-126413) into Sph I/Sal I digested pSP72 followed by insertion of a
the 695 bp BglII to BamHI fragment (nucleotides 131931 to 132626)
containing regions upstream of the ICP4 promoter including the viral
origin contained within the short inverted repeat regions into the BglII
to BamHI sites of the vector plasmid. The parental virus vHG was created
using the same targeting plasmid except that a HCMV-eGFP construct was
cloned into the same BamHI site resulting in the plasmid pSAE3. This
plasmid was recombined into an ICP4 and ICP27 deletion KOS strain HSV-1
mutant to produce parental vHG. For vHGlyRα1, candidate plaques
were initially selected for the loss of green fluorescence under
fluorescent microscopy. GlyR-HSV plaques were purified by three rounds of
limiting dilution with the GlyR construct verified by Southern blot
analysis. These vectors are schematically represented in FIG. 1.

[0043] Functional assay of channels in vitro. Human embryonic kidney (HEK)
293T cells were maintained as previously described (Qian et al., J.
Physiol., 562, 319-31 (2005)). For recordings, HEK 293T cells were plated
onto glass coverslips pretreated with poly-D-lysine (0.1 mg/ml) and
rat-tail collagen (0.1 mg/ml; BD Biosciences, San Jose, Calif.) in 35 mm
culture dishes at 1-2×105 cells per dish. 24 hours after
plating, the cells were infected with the GlyR-HSV construct. For
infections the growth medium was removed, the cells were washed twice
with serum free growth medium, and vHGlyRα1 was added at a
multiplicity of infection (MOI) of 10. The cells were then incubated at
37° C. for 1 hour. Following incubation, 1.5 ml of growth medium
(with serum) was added and the cells were incubated for an additional 24
hours at 37° C. Recordings were then performed 24 hours
post-infection at room temperature. Glycine-mediated whole-cell currents
were recorded with an Axopatch 200 amplifier (Molecular Devices, Union
City, Calif.) in voltage-clamp mode. Solutions were delivered via an
in-house fabricated fast perfusion system (Qian et al., J. Physiol., 538,
65-77 (2002)). The external solution contains (in mM): 140 NaCl, 5 KCl, 2
CaCl2, 1 MgCl2, and 10 HEPES; pH is 7.2+0.05, adjusted with
NaOH and osmolality 290+10 adjusted with sucrose as needed. The internal
solution contains (in mM): 140 CsCl, 1 CaCl2, 1 MgCl2, 2 MgATP,
10 HEPES, and 10 EGTA; pH is 7.3+0.05, adjusted with CsOH and osmolality
300+0.05. Data were recorded with a Digidata 1322A digitizer under the
control of pClamp 9.2 software (Molecular Devices) and all analyses were
performed with Clampfit 9.2 (Molecular Devices) or Origin 7.0 (OriginLab,
Northampton, Mass.). Percent desensitization was measured as
100×(1-ISS/Ipeak), where ISS and IPeak are the
amplitudes of current at steady state and peak, respectively.

[0045] Cell culture and histochemistry. In order to confirm expression in
neurons, dissociated primary doral root ganglia (DRG) neurons were
transduced with vHGlyRα1 and examined for glycine receptor
expression using immunofluorescent histochemistry. In this respect,
dorsal root ganglia (DRGs), which normally do not express GlyRs, from
17-day rat embryos were dissociated with 0.25% trypsin, 1 mM EDTA for 30
minutes at 37° C. with constant shaking and then plated on
poly-D-lysine-coated coverslips at 105 cells per well in 24-well
plates in 500 μl of defined Neurobasal medium containing B27, Glutamax
I, Albumax II, and penicillin/streptomycin (Invitrogen, Carlsbad,
Calif.), supplemented with 100 ng/ml of 7.0S NGF per ml (Sigma, St.
Louis, Mo.). Fifteen days following plating, cells were transduced with
either vHGlyRα1 or vHG at an MOI of 5 for one hour, washed with
fresh media, and incubated for an additional 24 hours. Cells were fixed
in 4% buffered formalin for 10 minutes, washed 3 times in PBS, and then
blocked in 5% normal goat serum, 0.2% tween-20, in PBS for 1 hour at room
temperature. Cells were incubated with a monoclonal antibody that
specifically recognizes the glycine receptor α-subunit (1:500; Cat#
146111, Synaptic Systems, Goettengen, Germany) overnight at 4° C.
Some cells were incubated without the primary antibody (primary delete).
Cells were washed 3 times in PBS then incubated with AlexaFluor 594
(1:1000, Invitrogen) for 1 hour at room temperature. Cells were washed 3
times in PBS, once in DI water, and mounted onto glass slides with Aqua
Poly/Mount (Poly sciences, Warrington, Pa.). Images were acquired on a
Zeiss Axiovert 200 microscope using an Axioncam MRCS high resolution
camera and Axiovision software (Zeiss, Thronwood, N.Y.).

[0047] Use of vHGlyRα1 to Treat Pain in a Rat Formalin Model. For in
vivo studies, rats were subcutaneously injected with vHGlyRα1 or
vHG, a control vector without α1 GlyR, into the plantar surface of
the right hind foot. Analgesic effects of viral injections were assessed
one week later by subcutaneous injection of formalin.

[0048] Specifically, 3-month-old male Sprague-Dawley rats (225-250 g) were
injected with 200 μl (1×108 plaque forming units) of either
vHGlyRα1 or vHG into the plantar surface of the right hind foot.
One week later the analgesic properties of the vectors was assessed by
subcutaneously injecting 50 μl of 2.5% formalin into the right hind
foot and assessing pain-related behaviors as previously described (Goss
et al., Gene Ther., 8, 551-6 (2001)). To examine the effects of
activation of expressed GlyR, 50 μl of 100 mM glycine was injected
into the plantar surface of the formalin-injected foot. Channels were
inhibited by injection of 50 μA of 10 mM strychnine, a specific GlyR
inhibitor. The beginning of second phase pain varied between animals from
20 to 40 minutes; this variation was independent of the vector used.
Glycine was therefore injected immediately after the 30, 40, or 50 minute
observation period and strychnine was injected immediately after the 60,
70, or 80 minute observation period. In order to present the data in a
clear manner, this variation was removed from FIG. 4 and all animal data
were presented with second phase pain beginning at 20 minutes.

[0049] Infection with vHGlyRα1 alone did not have any effect on pain
response as injection of formalin resulted in a typical biphasic
nociceptive response (FIG. 4A). Second phase pain was significantly
reduced in vHGlyRα1-infected animals following injection of glycine
at the 30 minute time point (FIG. 4B, black circles), an effect that
lasted for the duration of the study. The observed alleviation of pain
was reversed by injection of strychnine, a specific GlyR inhibitor, at 60
minutes (FIG. 4B, gray circles). Glycine application resulted in a small,
transient, non-significant dip in nociceptive behavior in vHG transfected
animals (FIG. 4B, open squares), suggesting that there may be some
endogenous glycine-dependent activity; strychnine had no effect in these
animals (data not shown). The robust response to added strychnine in
reversing the action of added glycine in vHGlyRα1-infected animals
strongly indicates that the pain alleviation is primarily due to
activation of expressed α1 GlyRs, and cannot be attributed to some
indirect effect (e.g., glycine acting as a co-agonist to NMDA receptors).

[0050] Use of vHGlyRα1 to Treat Pain in a Rat Osteosarcoma Model.
For the osteolytic sarcoma model, 6 week old C3H/HeJ mice had 105
NCTC 2472 osteolytic sarcoma cells implanted into the medullary space of
their right femur as previously described (Goss et al., Ann. Neurol.,
52(5): 662-5 (2002)). One week later, some groups of mice were injected
with 20 μl (2×107 pfu) of either vHGlyRα1 or vHG into
the plantar surface of the right hind foot; non tumor implanted mice and
tumor implanted mice that received no vector were used as controls. Two
weeks after vector injection mice were placed individually into a plastic
box and scored for pain-related behavior during open field motor activity
by an observer blinded to treatment group using a spontaneous ambulatory
pain score graded as: normal (0), slight limp while walking (1);
exaggerated limp (missing steps with the affected limb) (2); holding the
affected limb elevated while moving or stationary (3); biting and licking
the affected limb (4). Mechanical allodynia (MA) was then assessed using
an electronic von Frey anesthesiometer (IITC Life Sciences, Inc; Woodland
Hills, Calif.) fitted with a semiflexable tip. The next day, all mice
were injected with 20 μl of 100 mM glycine into the right footpad, and
15 minutes later spontaneous ambulatory pain and MA were re-assessed. All
of the animals treated with the glycine receptor vector demonstrated
decreased pain as assessed with both measurements.

[0051] This example demonstrates that the inventive method can be used to
treat pain in vivo.

[0054] Formalin footpad test (FFT): This is an acute model of inflammatory
pain (Dubisson and Dennis, Pain, 4(2): 161-74 (1977)). 50 μl of 2.5%
formalin will be injected subcutaneously into the plantar surface of the
right hind foot. This will cause acute pain lasting approximately 1-2
hours. Following formalin injection, each animal will be placed in a
clear rectangular plexiglass cage (20 cm wide×60 cm long×20
cm high). Different positions of the hind paw will be rated continuously
over a 3-minute period immediately after injection of the formalin and
every 10 minutes thereafter until the pain subsides completely. The
following scale will be used: 1=paw rests normally on floor; 2=paw rests
lightly on floor, toes ventroflexed; 3=whole paw is elevated; 4=animal
licks/bites paw. Weighted pain scores will be calculated by multiplying
the amount of time the rat spends in each position according to the
following formula: 0*t1+1*t2+2*t3+3*t4/180; where t1, t2, t3, and t4 are
the duration (in seconds) spent in categories 1, 2, 3, and 4
respectively. Rats will be euthanized at the end of the testing and
tissue removed for analysis. Sham animals will be injected with PBS.

[0055] Complete Freund's adjuvant test (CFA): This is a model of
inflammatory pain that better mimics the time course of postoperative
pain or other types of persistent injury (Iadarola et al, Brain, 121(Pt
5): 931-47 (1998)). Sprague-Dawley rats will be used in this model.
Baseline measurements of mechanical allodynia (MA) and thermal
hyperalgesia (TH) will be performed on all rats. 50 μl complete
Freund's adjuvant (CFA, undiluted) is injected subcutaneously into the
plantar surface of the right hind foot. Injection of CFA will cause an
inflammatory response resulting in both mechanical allodynia and thermal
hyperalgesia. This reaction usually occurs within 2 hours of injection of
CFA and peaks at 8-10 hrs; MA and TH can be assessed several times during
this initial phase. Hyperalgesia and edema usually last 1-2 weeks, during
which, MA and TH can be assessed.

[0056] Spinal nerve ligation: L5 spinal nerve ligation will be used to
model neuropathic pain in rats. All surgical instruments will be steam
heat sterilized prior to surgery. The instruments will be heat sterilized
using a hot bead benchtop sterilizer between animals in a given day. Rats
will be anesthetized with ketamine/xylazine (80/10 mg/kg) prior to
surgery. The dorsal surface will be palpated to locate the rostral
borders of the sacrum and dorsal spinous processes of the lower thoracic
and lumbar vertebrae, the T11-T12 laminae will be located by finding the
last rib, which attaches to the rostral end of the T13 vertebrae. The
hair overlying this area will be cleaned with 70% ethyl alcohol and the
surgical field will be shaved and prepared with three alternating scrubs
of Betadine and 70% alcohol, and a longitudinal incision will be made
exposing several segments of vertebrate. The left paraspinal muscles will
be separated from the spinous processes at the L4-S2 levels. The L6
transverse process will be removed with a small rongeur to identify
visually the L4-L6 spinal nerves. The left L5 spinal nerve will be
isolated and tightly ligated with 3-0 silk thread. The same procedure
will be performed on sham-operated animals except for the ligation.
Muscle and fascia will be sutured closed and the skin is closed with
autoclips, which will be removed after two weeks. The animals will be
allowed to recover on a 35° C. heating pad. Post operative plans
include penicillin as a prophylactic antibiotic.

[0057] Chronic constriction injury: Ligation of the sciatic nerve will be
used to model neuropathic pain in mice. All surgical instruments will be
steam heat sterilized prior to surgery. The instruments will be heat
sterilized using a hot bead benchtop sterilizer between animals in a
given day. The hair overlying the right thigh just lateral to the spinal
cord will be cleaned with 70% ethyl alcohol, and the surgical field will
be shaved and prepared with three alternating scrubs of Betadine and 70%
alcohol. A 1-2 cm incision will be made in the skin and the sciatic nerve
will be exposed at the level of the middle of the thigh by blunt
dissection through the biceps femoris. Proximal to the sciatic nerve's
trifurcation, about one cm long area of the sciatic nerve will be cleaned
of connective tissue and two ligatures (using 4.0 monofilament) will be
tied loosely around the nerve with about 3 mm between each ligation. The
ligatures will be tied such that the diameter of the nerve will be just
barely constricted when viewed by 30× magnification. This degree of
constriction will retard, but not arrest, the circulation through the
superficial epineural vasculature and may produce a small, brief twitch
in the muscle around the exposure. The incision will be closed in layers.
Sham operated control animals will undergo the same procedure but without
the sciatic nerve ligature.

[0058] Osteolytic sarcoma model of bone cancer pain: This is a pain model
that has characteristics of both neuropathic and inflammatory pain.
Behavioral testing will be performed following administration of HSV
vectors, and includes assaying spontaneous ambulatory pain, mechanical
allodynia, and thermal hyperalgesia as discussed below.

[0059] The ambulatory behavior of mice implanted with osteolytic sarcoma
cells can be graded to assess the degree of nociception. Mice will be
placed in a clear plastic 30×20 cm container and observed for
problems in spontaneous ambulation during a 2 minute period. The
following scores will be given to animals: 0 (normal), 1 (slight limp of
implanted limb), 2 (exaggerated limp with some guarding of implanted
limb), 3 (excessive guarding of implanted limb and failure to use
implanted limb while moving), and 4 (licking and biting of implanted
limb).

[0060] To assay mechanical allodynia, each animal will be placed into a
plexiglass enclosure (23×10 cm for rats, 10×10 cm for mice)
on a wire mesh elevated 20 cm above the benchtop. The mechanical
threshold will be measured by applying a series of von Frey hairs to the
midplantar surface of each hind paw. The von Frey hairs are a set of
calibrated filaments of increasing diameter and stiffness. As they are
pushed against the bottom of the paw, animals will either retract their
foot or not depending if the pressure from the von Frey hair is painful.
Withdrawal of the hind paw within 5 seconds will be deemed a positive
response; no paw withdrawal will be deemed a negative response. The
up-down method will be utilized to determine the 50% gram threshold
(Chaplan et al., J. Neurosci. Methods, 53(1): 55-63 (1994), and Chaplan
et al., J. Pharmacol. Exp. Ther., 269(3): 1117-1123 (1994)). In this
respect, a positive response is followed by application of the next grade
higher filament and a negative response is followed by presentation of
the next grade lower filament; this continues until the 50% gram
threshold response is determined.

[0061] Thermal hyperalgesia (TH) will be determined using a plantar
analgesia meter (IITC Life Sciences, Woodland Hills, Calif.). Each animal
will be placed into a plexiglass enclosure and placed on a glass surface
maintained at 30° C. After a 15 minute accommodation period, a
light beam will be focused onto the midplantar area of each hind paw and
the amount of time it takes the animal to move its paw from the heat
source will be measured. The heat source automatically turns off if the
animal does not respond in 20 seconds in order to ensure that no tissue
damage will occur. Three trials will be performed for each side with a 15
minute period between each trial and the times will be averaged.

[0062] HSV vector footpad inoculation: Animals will be anesthetized with
Isoflurane. Paper towels will be placed in the bottom of a desiccator jar
and a small volume of Isoflurane (0.3 ml per 0.5 L of desiccator space)
will be poured onto the towels. A ceramic floor with holes will be placed
over the towels and the animals will be placed into the jar with the lid
on top until they stop moving. Animals will be removed; a tail and foot
pinch will be performed to make sure the animals are fully anesthetized.
100 μl (rats) or 20 μl (mice) of HSV vector (vHGlyRα1 or vHG)
will be injected subcutaneously into the midplantar region of the foot
using a sterile 0.5 cc insulin syringe with a 30 g attached needle.
Animals will be placed back into their cage and allowed to recover (about
5 minutes). This procedure will be performed under a chemical fume hood.

[0063] Identification of vHGlyRα1-Infected Cells: Infected neurons
will be identified using a Red gene labeled vector, and Cy5-labeled
strychnine will be used to determine the distribution of the GlyR. The
response to anesthetics that are known agonists of the GlyR will be
assayed to demonstrate their relative activity and effects on nerve
conduction. Further nerve conduction studies will be carried out using
rat nerve fiber explants infected with the GlyR vector to confirm the
activity of GlyR in single neurons. In dose finding and repeat dosing
experiments, the method for maximum vector uptake will be determined. The
presence of vector will be determined by fluorescence analysis of tissue
sections including the sensory ganglia. The vector genome copy number
will be determined by quantitative DNA PCR analysis of infected tissue,
and the expression of GlyR gene expression will be evaluated by
quantitative RT-PCR. The kinetics of GlyR gene expression will be
determined when under the control of a more transient promoter (e.g.,
HCMV) and the long-term promoter (LAP2), a component of the viral latency
gene promoter. The co-localization of Red gene expression and
immuocytochemistry studies will be used to determine the type of
neuron(s) harboring the HSV vector.

Example 3

[0064] This example demonstrates a method of generating a transgenic
animal that exogenously expresses a GlyR protein.

[0065] A cb-stop-lacZ construct (Zinyk et al., Curr Biol., 8(11):665-8
(1998)) will serve as the base construct for production of transgenic
mice. This construct contains (5' to 3') the constitutively active
chicken β-actin promoter and the first two non-coding exons, a
transcription/translation stop cassette that is flanked by loxP
sequences, a lacZ reporter cassette, and the 3' untranslated
region/polyadenylation site from the mouse protamine-1 gene. In previous
studies of transgenic mice that harbor this construct (see, e.g., Zinyk
et al., supra), it was demonstrated that in the absence of Cre-mediated
recombination, the reporter cassette was inactive. Following Cre-mediated
recombination and deletion of the stop cassette, the reporter cassette
was expressed.

[0066] Standard molecular techniques will be used to replace the lacZ
reporter with either a wild-type or a mutant (M287L) mouse glycine
α1 receptor coding sequence. The M287L mutation results in greatly
enhanced sensitivity to the volatile anesthetic enflurane. The transgene
inserts will be isolated from the plasmid backbone following digestion
with Sal1-Asp718, and transgenic mice will be made using standard
transgenic pronuclear microinjection techniques at the University of
Pittsburgh Transgenic and Gene Targeting Core Facility. C57BL/6J zygotes
will be used for pronuclear microinjections. Transgenic founders will be
identified by Southern blot or PCR analysis of tail DNA and subsequently
bred to C57BL/6J mice to establish the transgenic lines.

[0067] To create mice that selectively express a glycine receptor
transgene in sensory neurons, transgenic mice will be mated to "knock-in"
mice in which Cre recombinase was inserted into the Nav1.8 locus
(Stirling et al., Pain, 113(1-2): 27-36 (2005)). These mice effectively
and selectively induce recombination only in sensory neurons (Stirling et
al., supra). Mice that harbor both the glycine receptor and the
Nav1.8-Cre transgenes will be compared to nontransgenic controls as well
as to each single transgenic line.

[0068] Transgene expression and functionality will be analyzed by assaying
(1) the distribution of HSV-GlyR in afferent populations, (2) the
localization of HSV-GlyR within sensory neurons, (3) the activity of
HSV-delivered GlyR protein in transduced afferent populations, (4) the
duration of functional GlyR expression, and (5) the functional effects of
GlyR activation as a function of the pain model tested. Double and triple
label immunohistochemistry will be used to characterize the population of
afferents infected with HSV as well as the infection efficiency.
Populations to be studied can be broken down into the following
categories: (a) myelinated versus unmyelinated using NF200 and
peripherin, (b) trophic factor receptor maintaining phenotype: TrkA
(small peptidergic), TrkC, GFR-alpha1, GFR-alpha2 and GFR-alpha3, (c)
peptide content: CGRP and IB4, and (d) nociceptive signaling molecules:
TRPV1, TRPA1, NaV1.8, P2X3. The efficiency of labeling will be assessed
with the use of retrograde tracers co-injected with HSV. With this
approach, the percentage of each type of afferent innervating the site of
injection can be determined.

[0069] The distribution of functional GlyR within a sensory neuron
relative to sites of sensory transduction, action potential generation,
action potential propagation, and transmitter release will be assayed
using a combination of traditional immunohistochemistry and receptor
binding.

[0070] To determine the activity of HSV-delivered GlyR protein in
transduced afferent populations, a series of electrophysiological studies
will be performed on isolated sensory neurons and the isolated sciatic
nerve. The isolated sensory neurons will enable characterization of
afferent subpopulations in which cutaneous HSV infection results in the
generation of functional receptors. The isolated axon preparation will
enable determination of whether or not receptors present in mid axons are
functional through their ability to block propagated action potentials. A
skin-nerve preparation also will be used to determine whether functional
receptors are present in afferent terminals.

[0071] The duration of functional GlyR expression will be determined using
a series of behavioral pharmacological and anatomical approaches.
Behavioral experiments will indicate the duration over which functional
receptors are present and whether receptor activation influences the
duration of receptor expression. The anatomical experiments will be used
to assess the time frame over which GlyR and HSV are detected following
inoculation.

[0072] Incorporated by reference are all references, including
publications, patent applications, and patents, cited herein to the same
extent as if each reference were individually and specifically indicated
to be incorporated by reference and were set forth in its entirety
herein.

[0073] The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the singular and
the plural, unless otherwise indicated herein or clearly contradicted by
context. The terms "comprising," "having," "including," and "containing"
are to be construed as open-ended terms (i.e., meaning "including, but
not limited to,") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range, unless
otherwise indicated herein, and each separate value is incorporated into
the specification as if it were individually recited herein. All methods
described herein can be performed in any suitable order unless otherwise
indicated herein or otherwise clearly contradicted by context. The use of
any and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the invention and does
not pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of the
invention.

[0074] Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become apparent
to those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the invention to
be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described elements
in all possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by context.

Patent applications by David Krisky, Sewickley, PA US

Patent applications by James R. Goss, Bethel Park, PA US

Patent applications by Joseph C. Glorioso, Iii, Blawnox, PA US

Patent applications by Michael Cascio, Pittsburgh, PA US

Patent applications by University of Pittsburgh - Of the Commonwealth System of Higher Education